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MELNHE Overview

Although temperate forests are generally thought of as N-limited, resource optimization theory predicts that ecosystem productivity should be co-limited by multiple nutrients. These ideas are represented in the Multi-Element Limitation (MEL) model (Rastetter et al. 2012). To test the patterns of resource limitation predicted by MEL, we are conducting nutrient manipulations in three study sites in New Hampshire: Bartlett Experimental Forest, Hubbard Brook Experimental Forest, and Jeffers Brook in the White Mountain National Forest.

Site Description

Sampling Design

In 2010 (pretreatment), we established 4 soil sampling subplots in each plot in order to confine the disturbance associated with sampling and at the same time ensure that repeated sampling was always done in the same general area. In 2014 and 2017 3-4 soil cores were collected in each soil sampling subplot and divided into horizons (Oe, Oa, 10 cm of mineral). All cores in one plot (12-16 total, from the 4 subplots) were mixed together within each horizon and homogenized for one composite sample per horizon per plot.

Laboratory Procedures

Samples were refrigerated until processing, and were processed within 4 days of collection (usually processed the day after collection). Samples were homogenized by gentle separation of organic fragments over a 4-mm sieve followed by thorough mixing. Fragments > 4 mm were not included. Soil moisture content was determined by drying subsamples at 60 degrees C to constant mass. Soils were then stored at -20 °C before enzyme assay. Soil extracellular enzyme activity was measured using 96-well plate based fluorescent and colorimetric methods. A soil subsample was ground in 50 mM sodium acetate buffer (pH 4.5) with a food blender to produce a soil slurry. The soil slurry was pipetted into a 96-well microplate carrying fluorescent groups linked substrates and standards for the measurement of Leucine amino-peptidase, alanine aminopeptidase, β-N-acetylglucosaminidase, β-glucosidase, cellobiohydrolase, and acid phosphatase. Polyphenol oxidase activity was measured colorimetrically by oxidation of L-3,4-dihydroxyphenylalanine. We also measured the Michaelis-Menten kinetics of alanine amino-peptidase, β-N-acetylglucosaminidase, and acid phosphatase. The maximal rate of velocity (Vmax) and half-saturation constant (Km) of these enzymes were obtained using substrate concentration series for alanine aminopeptidase (100, 200, 500, 750, 1000, 2000 µM), β-N-acetylglucosaminidase (100, 200, 300, 500, 750, 1000 µM), and acid phosphatase (100, 200, 500, 750, 1000, 1400, 1800, 2500 µM).

Calculations

Enzyme activities were obtained by comparing the fluorescent reading of a sample assay to the standard curve, expressed in the unit of µmol g-1 h-1. The Michaelis-Menten maximal velocity (Vmax) and half-saturation constant (Km) were estimated for each enzyme using non-linear regression following the simple Michaelis-Menten model:

V=(Vmax[S]/(Km+[S])

Where V is the reaction rate, [S] is the substrate concentration, Vmax is the maximal reaction rate, and Km is the substrate concentration at half- maximal reaction rate or when half of the active sites of the enzyme are filled. The regression analyses were conducted using the curve-fitting program of GraphPad Prism (GraphPad software, San Diego, CA).